Mitochondria, Antioxidants and Aging

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Mitochondria, Antioxidants and Aging
Holly Van Remmen, Ph.D. GRECC , South Texas
Veterans Health Care System Department of
Cellular and Structural Biology Barshop Institute
for Longevity and Aging Studies University of
Texas Health Science Center San Antonio
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Review of the Role of Mitochondria in Aging

• Mitochondrial Function and Oxidative Stress
• Mitochondrial Theory of Aging
• Studies Manipulating Mitochondrial Oxidative
  Stress in Animal Models-what can we learn?

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ATP Production
Electron Transport Chain
I
III
IV
II
ATP Synthase
H2O
O2
ATP
NADH
TCA Cycle
FADH
Fatty Acid Oxidation
Ca Regulation
Signaling
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Sources of Reactive Oxygen Species
Non mitochondrial NADPH Oxidases Microsomal
cytochrome P-450 Cyclooxygenases Monoamine
oxidases Peroxisomal b oxidation of fatty
acids Phagocytes
gt90 is mitochondrial electron transport chain
contains several redox centers that may leak
electrons to oxygen
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H2O2
GPX
cytoplasm
CAT
SOD1
O2
H2O2
O2?
Intermembrane space
III
GPX
H2O2
Matrix
SOD2
O2
O2?
Mitochondrial ROS generation
ROS production is at Complex I and Complex III
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Mitochondrial Antioxidant Defense
CuZnSOD
ATP
V
III
II
IV
I
H2O2
O2-
H2O2
Trx2
MnSOD
Peroxisomes
GPx
H2O
PrxIII
H2O2
Catalase
H2O
Trx1

O2
H2O2
O2-
Nucleus
2H2O
CuZnSOD
GPx
nDNA
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Consequences of mitochondrial oxidative stress
Marker
F2-Isoprostanes
8-iso-PGF2a
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The problem Mitochondria are required for
energy production
Mitochondria produce potentially harmful reactive
oxygen species
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mitochondria
What is the link?
AGING
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Some Current Theories of Aging

• Rate of living theory
• Energy consumption is limiting for longevity
• Evolutionary Theories
• Aging is a programmed event
• Mutation accumulation
• Antagonistic Pleiotropy
• Genomic Instability
• Cell Senescence/Telomere Shortening
• GH/IGF-1 axis
• Free Radical Theory

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Free Radical Theory of Aging
Aging a theory based on free radical and
radiation chemistry 1956, D. Harman
J.Gerontology 11(3)298-300
A single common process, modified by genetics and
environmental factors, is responsible for the
aging and death of all living things.
Aging and the degenerative diseases associated
with it are attributed basically to the
deleterious side attacks of free radicals on cell
constituents and on the connective tissues..
The free radicals probably arise largely through
reactions involving molecular oxygen catalyzed in
the cell by oxidative enzymes and in the
connective tissues by traces of metals such as
iron, cobalt, and manganese."
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Oxidative Stress Theory of Aging
Mitochondrial (ETC)
Extra-mitochondrial (NADPH oxidase)
Exogenous Sources (chemicals,radiation)
ROS
NO
Imbalance between pro-oxidants and antioxidants.
O2-
H2O2
OH.
ONOO-
Chronic state of oxidative stress
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Oxidative Stress Theory of Aging
Mitochondrial (ETC)
Extra-mitochondrial (NADPH oxidase)
Exogenous Sources (chemicals,radiation)
Steady-state accumulation of oxidative damage
that increases during aging
ROS
NO
O2-
H2O2
Antioxidant Defense
ONOO-
OH.
Progressive loss in efficiency of cellular
processes
Net Result
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Mitochondria are major source of ROS
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Mitochondrial Theory of Aging
Harman,D. 1972 The biologic clock the
mitochondria? J. Am Ger Society 20145-57 Free
radicals escaping from the respiratory chain
would be expected to produce deleterious effects
mainly in the mitochondria Are these effects
mediated in part by mitochondrial DNA functions?
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Mitochondria Theory of Aging
Increased Production of ROS
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Mitochondria Theory of Aging
-Alterations in ETC function -mtDNA
deletions/mutations
Increased Production of ROS
Damaged mitochondria produce more ROS
Accumulation of oxidative damage with age
Decreased ATP Cell Death/Apoptosis Functional
Decline
Aging
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Early studies supported an increase in ROS with
age
Housefly flight muscle (submitochondrial
particles) Sohal and Sohal (1991) Mech of
Aging and Dev. 187-202.
Mouse kidney, heart and brain mitochondria (submi
tochondrial particles) Sohal et al
(1994) Mech.Ageing Dev.121-133
Rat vastus lateralis or soleus
muscle (homogenates/isolated mitochondria) Bej
ma and .Ji (1999) J.Appl.Physiol. 465-470
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Other studies show no increase..
Rat Muscle
Capel et al. Soleus Mech. Aging Dev.,
(2004) 367-373. Tibialis Anterior Drew et
al Am.J.Physiol Gastrocnemius Regul.Inte
gr.Comp Physiol, (2003) R474-R480
Rat Heart
Hansford et al J.Bioenerg.Biomembr., (1997)
89-95
Rat Liver
Lopez-Torres et al. Free Radic.Biol.Med.,
(2002) 882-889
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Increase may be tissue specific?.....
Mouse hindlimb Skeletal muscle
Mouse heart
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Mitochondria Theory of Aging
Increased Production of ROS
Some tissues?
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Aging and ETC function
Complex I II III
IV V
Muscle Tissue
Human muscle
Trounce et al., (1989) Lancet (8639)637-9
Human muscle
Boffoli et al. (1994) BBA 122673-82
Sugiyama et al. (1993) Biochem Mol Biol Int
30937-44
Rat muscle
Muller-Hocker et al. (1990) J Neurol Sci
10014-21
Human muscle
Desai et al. (1996) Arch Bioc Biophys
333145-151
B6C3F1 mice
Muller-Hocker et al. (1996) Mech Aging Dev
86197-213
monkeys
Kwong et al. (2000) Arch Bioc Biophys 373 16-22
C57Bl6 mice
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More recent studies in humans, mice
Barrientos 1996 Rasmussne 2003 Chretien
1998, Kwong and Sohal 2000
No consistent change
Current evidence suggests that there is no
consistent overall defect in the ETC with age.
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Mitochondria Theory of Aging
-Alterations in ETC function (?)
-mtDNA mutations/deletions
Increased Production of ROS
Some tissues?
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MtDNA
16549 bp
-Double-stranded, circular molecule -2 to 10
copies in each mitochondrion gt 1,000 in each
cell
mtDNA encodes for 37 genes 22tRNA 13
mitochondrial peptides (ETC subunits) 16S and 26S
rRNA
(Remaining gt 67 ETC subunits are nuclear encoded)
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mtDNA is more sensitive to oxidative damage than
nuclear DNA
Estimated at 1/8000 bases for mtDNA vs 1/130,000
for nDNA (Richter et al 1988 PNAS)
-high metabolism -proximity to generation of
ROS -lack of histones -repair systems less
efficient
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mtDNA Deletions

• Tissues that turn-over more slowly (skeletal
  muscle, heart) have more
• mtDNA deletions than more rapidly dividing
  tissues (liver)

• Deletions increase with age in muscle heart and
  brain (identified using PCR)

• Common deletion mtDNA4977 increases by a factor
  of 10,000
• in muscle during a normal human lifespan
• reaching 0.1 of total muscle mtDNA by 84 yrs

• Evidence indicates focal accumulation of
  deletions in some tissues

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Mitochondrial Theory of Aging
Alterations in ETC function (?) mtDNA
mutations/deletions YES
Increased Production of ROS
Some tissues?
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Age related changes in mitochondrial DNA
oxidative damage
Liver Mitochondrial DNA
Liver Nuclear DNA
0.8
2.5
0.6
2.0
oxo8dG/2dG x105
0.4
1.0
0.2
0.0
0.0
3 6 months
26 months
3 6 months
26 months
Van Remmen et al., Physiol. Genomics, 2003
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Plasma Free Isoprostanes
Carbonyl Groups in Liver Protein
0.12
200
0.10
150
0.08
Carbonyl Levels (U/mg protein)
0.06
8-isoprostane (pg/ml)
100
0.04
50
0.02
0
0
Young (4-6 mths) Old (24-28 mths)
Young (10 mths) Old (28-29 mths)
Asish Chaudhuri, San Antonio
Jack Roberts, Vanderbilt University
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Mitochondrial Theory of Aging
Alterations in ETC function (?) mtDNA
mutations/deletions YES
Increased Production of ROS
Damaged mitochondria produce more ROS
Some tissues?
Accumulation of oxidative damage with
age (mitochondrial and non-mitochondrial) YES
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Effects of Manipulating Mitochondrial Oxidative
Stress in Animal Models
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Altered expression of MnSOD in Drosophila
Sun et al., Genetics. (2002) 161(2)661-72.
Induced overexpression of mitochondrial Mn-SOD
extends the life span of adult Drosophila
melanogaster.
Mean life span increased by an average of 16
Another study finds no increase in lifespan in
MnSOD Tg flies (Orr et al., 2003 JBC)
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Increased Expression of Mitochondrial Uncoupling
Protein 2
Targeted expression of the human uncoupling
protein 2 (hUCP2) to adult neurons extends life
span in the fly.
Alterations in Mitochondrial Function and
Oxidative Stress

• decrease in ROS production
• decrease in oxidative damage
• increased resistance to paraquat

Increase in lifespan
Fridell et al., Cell Metab. 2005 1(2)145-52
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Increased expression of Mitochondrial genes
Effect on Lifespan
MnSOD
UCP2
Seem to be consistent with the theory..
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What can we learn about mitochondrial theory of
aging from mouse models?

• MnSOD-mitochondrial antioxidant
• mtDNA polymerase mutant mice
• Mitochondrial targeting of catalase

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MnSod (Sod2/-) Knockout mice

• Mitochondrial antioxidant enzyme (O2- O2-
  H2O2 )
• Homozygous mutant is lethal (survival is less
  than 2 weeks)

• 50 decrease in MnSOD activity in all tissues
  studied

• No compensation by other major antioxidant enzymes

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Wt
Sod2 /-
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MnSOD Activity (relative units)
10
0
(Li et al. (1995) Nature Genetics.
muscle
brain
kidney
heart
spleen
liver
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Sod2/- Knockout Mice
CuZnSOD
H2O2
ATP
II
V
III
I
IV
O2-
X
H2O2
Trx2
Peroxisomes
MnSOD
GPx
GPx
b -Oxidation pathway
H2O
2H2O
PrxIII
H2O2
Catalase
H2O

Trx1
O2
H2O2
O2-
2H2O
CuZnSOD
GPx
2GSH
GSSG
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Mitochondrial Theory of Aging
Alterations in ETC function mtDNA
mutations/deletions
Increased Production of ROS
Sod2/- mice (Reduced Antioxidant Protection)
Decreased ATP Cell Death Functional Decline
Increased Aging
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MnSOD Heterozygous Knockout Mice
Oxidative damage to specific mitochondrial
proteins aconitase NADH oxidoreductase
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MnSOD Heterozygous Knockout Mice
Altered Mitochondrial Function /Decreased
activities of ETCs
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MnSOD Heterozygous Knockout Mice
Increased oxidative damage to DNA
Nuclear DNA
6 month
26-28 month
c
0.8
0.6
oxo8dG/2dG x105
0.4
0.2
0.0
WT
Sod2/-
WT
Sod2/-
WT
Sod2/-
WT
Sod2/-
Heart
Muscle
Liver
Brain
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Mitochondrial Theory of Aging
Alterations in ETC function mtDNA
mutations/deletions
Increased Production of ROS
Sod2/- mice (Reduced Antioxidant Protection)
Aging
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MnSOD Heterozygous Knockout Mice
No change in survival
Increase in Cancer
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Premature ageing in mice expressing defective
mitochondrial DNA polymerase
Nature, May 27, 2004, Trifunovic et al
Homozygous knock-in mice for a proof reading
deficient PolgA-mtDNA polymerase

• 3-5 fold increase in point mutations and
  deletions in mtDNA
• Normal appearance until 6 months (25 weeks)
• -Weight loss, kyphosis, alopecia, decreased
  fat,osteoporosis
• Decreased ETC activity and ATP production in
  heart

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mtDNA mutations -- Shortened lifespan

• Median lifespan, 48 weeks, or 336 days
• All died by 61 weeks, or 430 days

a link between mtDNA mutations and ageing
phenotypes in mammals?
PolgA-mtDNA polymerase mutants
Nature, May 27, 2004, Trifunovic et al
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Mitochondrial DNA Mutations, Oxidative Stress,
and Apoptosis in Mammalian Aging
Kujoth et al Science 259, 2005
-Targeted mutation of PolgA Mitochondrial DNA
polymerase -Residue substitution in the
exonuclease domain that impairs proof reading
ability

• increased mutations in mtDNA

• No increase in oxidative damage

• Apoptotic markers were increased during aging
• increased caspase 3 activity

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Phenotype shows age-associated characteristics
-Indistinguishable in young age -Phenotype
evident at 9 months

• Hair loss, graying, and kyphosis.
• Thymic involution
• Testicular atrophy associated with the depletion
  of spermatogonia
• Loss of bone mass
• Loss of intestinal crypts
• Decrease in circulating red blood cells
• Weight loss
• Hearing loss
• Loss of muscle mass

Kujoth et al Science 259, 2005
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• Maximal survival, 460 days.
• Median survival, 416 days
• Wildtype, both are gt 850 days

Are mtDNA mutations linked to apoptosis and
aging?
Mitochondrial DNA polymerase mutant
Kujoth et al Science 259, 2005
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Extension of Murine Life Span by Overexpression
of Catalase Targeted to Mitochondria
CATALASE
Increased expression in heart muscle and brain
Increased over 50x in heart mitochondria
Science, 2005- Schriner et al.
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17 21 INCREASE IN LIFESPAN
Both median and maximum lifespan were increased
in MCAT animals
Science, 2005- Schriner et al.
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• Cardiac pathology and cataract development were
  delayed
• H2O2 production was reduced
• Oxidative damage was reduced (aconitase activity,
  8-OHdG)
• The development of mitochondrial deletions was
  reduced.

Support for the free radical theory of aging and
the importance of mitochondria ??
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Lifespan Effect on Lifespan

• 50 reduction in MnSOD
• Increased mtDNA mutations
• Mitochondrial targeting of catalase

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Do mitochondria play a key role in aging??
Evidence to date would suggest that mitochondria
and oxidative stress in general certainly play a
role in age related disease, but a direct link
between mitochondria and aging has not been
definitively established